Synthesis and Identification of Biologically Active Mono-Labelled FITC-Insulin Conjugate.

Fluorescently labelling proteins such as insulin have wide ranging applications in a pharmaceutical research and drug delivery. Human insulin (Actrapid®) was labelled with fluorescein isothiocyanate (FITC) and the synthesised conjugate identified using reverse phase high performance liquid chromatography (RP-HPLC) on a C18 column and a gradient method with mobile phase A containing 0.1% trifluoroacetic acid (TFA) in Millipore water and mobile phase B containing 90% Acetonitrile, 10% Millipore water and 0.1% TFA. Syntheses were carried out at varying reaction times between 4 and 20 h. Mono-labelled FITC-insulin conjugate was successfully synthesised with labelling at the B1 position on the insulin chain using a molar ratio of 2:1 (FITC:insulin) at a reaction time of 18 h and confirmed by electrospray mass spectroscopy. Reactions were studied across a pH range of 7-9.8 and the quantities switch from mono-labelled to di-labelled FITC-insulin conjugates at a reaction time of 2 h (2:1 molar ratio) at pH > 8. The conjugates isolated from the studies had biological activities in comparison to native insulin of 99.5% monoB1, 78% monoA1, 51% diA1B1 and 0.06% triA1B1B29 in HUVEC cells by examining AKT phosphorylation levels. MonoB1 FITC-insulin conjugate was also compared to native insulin by examining cell surface GLUT4 in C2C12 skeletal muscle cells. No significant difference in the cellular response was observed for monoB1 produced in-house compared to native insulin. Therefore mono-labelled FITC-insulin at the B1 position showed similar biological activity as native insulin and can potentially be used for future biomedical applications.

Electromembrane Extraction of Unconjugated Fluorescein Isothiocyanate from Solutions of Labeled Proteins Prior to Flow Induced Dispersion Analysis.

In this initial research on feasibility, removal of unconjugated fluorescein isothiocyanate (FITC) after fluorescent labeling of human serum albumin (HSA) with electromembrane extraction (EME) was investigated for the first time. A 100 µL solution of 0.1 mg/mL HSA was fluorescently labeled with 0.01 mg/mL FITC in a molar ratio of 10:1 in an Eppendorf tube for 30 min under agitation and absence of light. Then the labeled solution was transferred to a 96-well EME with 3 µL 0.1% (w/w) Aliquat 336 in 1-octanol as the supported liquid membrane (SLM) and 200 µL 10 mM NaOH as waste solution. EME was performed for 10 min with a voltage of 50 V, with the anode in the waste solution and at 900 rpm agitation. Negatively charged and unconjugated FITC was extracted electrokinetically into the SLM and to the waste solution. Analysis of purified samples, by Taylor dispersion analysis (TDA), showed a 92% removal of unconjugated FITC (FITC clearance: 92%, RSD: 3%), while 79% of the HSA/FITC complex remained in the sample (protein retention: 79%, RSD: 18%). Conserved functionality of the HSA/FITC complex after EME was proven by a binding affinity study with anti-HSA using flow induced dispersion analysis (FIDA). In this real sample, the dissociation constant (Kd) and hydrodynamic radius of the complex were determined to be 0.8 µM and 5.87 nm, respectively, which was in concordance with previously reported values.

Synthesis and Identification of FITC-Insulin Conjugates Produced Using Human Insulin and Insulin Analogues for Biomedical Applications.

Human insulin was fluorescently labelled with fluorescein isothiocyanate (FITC) and the conjugate species produced were identified using high performance liquid chromatography and electrospray mass spectroscopy. Mono-labelled FITC-insulin conjugate (A1 or B1) was successfully produced using human insulin at short reaction times (up to 5 h) however the product always contained some unlabelled native human insulin. As the reaction time was increased over 45 h, no unlabelled native human insulin was present and more di-labelled FITC-insulin conjugate (A1B1) was produced than mono-labelled conjugate with the appearance of tri-labelled conjugate (A1B1B29) after 20 h reaction time. The quantities switch from mono-labelled to di-labelled FITC-insulin conjugate between reaction times 9 and 20 h. In the presence of phenol or m-cresol, there appears to be a 10 % decrease in the amount of mono-labelled conjugate and an increase in di-labelled conjugate produced at lower reaction times. Clinically used insulin analogues present in commercially available preparations were successfully fluorescently labelled for future biomedical applications.

Preparation and characterization of a thermoresponsive gigaporous medium for high-speed protein chromatography.

A high-speed thermoresponsive medium was developed by grafting poly(N-isopropylacrylamide-co-butyl methacrylate) (P(NIPAM-co-BMA)) brushes onto gigaporous polystyrene (PS) microspheres via surface-initiated atom transfer radical polymerization (ATRP) technique, which has strong mechanical strength, good chemical stability and high mass transfer rate for biomacromolecules. The gigaporous structure, surface chemical composition, static protein adsorption, and thermoresponsive chromatographic properties of prepared medium (PS-P(NIPAM-co-BMA)) were characterized in detail. Results showed that the PS microspheres were successfully grafted with P(NIPAM-co-BMA) brushes and that the gigaporous structure was robustly maintained. After grafting, the nonspecific adsorption of proteins on PS microspheres was greatly reduced. A column packed with PS-P(NIPAM-co-BMA) exhibited low backpressure and significant thermo-responsibility. By simply changing the column temperature, it was able to separate three model proteins at the mobile phase velocity up to 2167 cm h(-1). In conclusion, the thermoresponsive polymer brushes grafted gigaporous PS microspheres prepared by ATRP are very promising in 'green' high-speed preparative protein chromatography.

Coating gigaporous polystyrene microspheres with cross-linked poly(vinyl alcohol) hydrogel as a rapid protein chromatography matrix.

Gigaporous polystyrene (PS) microspheres were hydrophilized by in situ polymerization to give a stable cross-linked poly(vinyl alcohol) (PVA) hydrogel coating, which can shield proteins from the hydrophobic PS surface underneath. The amination of microspheres (PS-NH2) was first carried out through acetylization, oximation and reduction, and then 4,4'-azobis (4-cyanovaleric acid) (ACV), a polymerization initiator, was covalently immobilized on PS-NH2 through amide bond formation, and the cross-linked poly(vinyl acetate) (PVAc) was prepared by radical polymerization at the surfaces of ACV-immobilized PS microspheres (PS-ACV). Finally, the cross-linked PVA hydrogel coated gigaporous PS microspheres (PS-PVA) was easily achieved through alcoholysis of PVAc. Results suggested that the PS microspheres were effectively coated with cross-linked PVA hydrogel, where the gigaporrous structure remained under optimal conditions. After hydrophilic modification (PS-PVA), the protein-resistant ability of microspheres was greatly improved. The hydroxyl-rich PS-PVA surface can be easily derivatized by classical chemical methods. Performance advantages of the PS-PVA column in flow experiment include good permeability, low backpressure, and mechanical stability. These results indicated that PS-PVA should be promising in rapid protein chromatography.

Nanoporous membranes enable concentration and transport in fully wet paper-based assays.

Low-cost paper-based assays are emerging as the platform for diagnostics worldwide. Paper does not, however, readily enable advanced functionality required for complex diagnostics, such as analyte concentration and controlled analyte transport. That is, after the initial wetting, no further analyte manipulation is possible. Here, we demonstrate active concentration and transport of analytes in fully wet paper-based assays by leveraging nanoporous material (mean pore diameter ≈ 4 nm) and ion concentration polarization. Two classes of devices are developed, an external stamp-like device with the nanoporous material separate from the paper-based assay, and an in-paper device patterned with the nanoporous material. Experimental results demonstrate up to 40-fold concentration of a fluorescent tracer in fully wet paper, and directional transport of the tracer over centimeters with efficiencies up to 96%. In-paper devices are applied to concentrate protein and colored dye, extending their limits of detection from ∼10 to ∼2 pmol/mL and from ∼40 to ∼10 µM, respectively. This approach is demonstrated in nitrocellulose membrane as well as paper, and the added cost of the nanoporous material is very low at ∼0.015 USD per device. The result is a major advance in analyte concentration and manipulation for the growing field of low-cost paper-based assays.

Improved high-speed capillary electrophoresis system using a short capillary and picoliter-scale translational spontaneous injection.

Here, we describe an improved high-speed CE (HSCE) system using a short capillary and translational spontaneous sample injection. Several important factors for consideration in system design as well as various factors influencing the performance of the HSCE system were investigated in detail. The performance of this HSCE system was demonstrated in electrophoretic separation of FITC-labeled amino acids. Under optimized conditions, baseline separation of eight amino acids and FITC were achieved in 21 s with the plate heights ranging from 0.20 to 0.31 µm, corresponding to a separation rate up to 20 700 theoretical plates per second. The separation speed and efficiency of the optimized high-speed CE system are comparable to or even better than those reported in microchip-based CE systems.

Multi-dimension microchip-capillary electrophoresis device for determination of functional proteins in infant milk formula.

To improve resolution of important minor proteins and eliminate time-consuming precipitation of major protein with associated analyte co-precipitation risk, a multi-dimension strategy is adopted in the 2D microchip-CE device to isolate major proteins on-chip, enrich minor proteins in capillary before their separation in CE for UV quantitation. A standard fluorescent protein mixture containing FITC-BSA, myoglobin and cytochrome as specific pI markers has prepared to demonstrate capability of the device to fractionate minor proteins by IEF. The results using a standard protein mixture with profile resembling infant milk formula show a complete isolation of high abundance proteins by a 2-min 1D IEF run. The subsequent t-ITP/CZE run by on-chip high voltage switching delivers a high stacking ratio, realizing 60 folds enrichment of isolated protein fractions. All five important functional proteins (LF, IgG, α-LA, ß-LgA and ß-LgB) known to fortify infant milk formula are isolated and determined using two consecutive t-ITP-CZE runs within a 18-min total assay time, a significant saving compared to several hours conventional pretreatment. For a 100g infant milk formula sample, working ranges of 20-8000mg, repeatability 3.8-5.3% and detection limits 2.3-10mg have been achieved to meet government regulations. Method reliability is established by 100% recoveries and agreeable results within expected ranges and labeled values. The capability of the device for field operation, rapid assay with quick results, label-free universal detection, simple operation by aqueous dissolution before injection, and the demanding matching in 2D separation based on isolated fractions at specified pI ranges, closely matched migration time and baseline-resolved peak shape makes the device a general tool to detect unknown proteins and determine known minor proteins in protein-rich samples with interfering constituents.

Ion concentration polarization in a single and open microchannel induced by a surface-patterned perm-selective film.

We describe a novel and simple mechanism for inducing ion concentration polarization (ICP) using a surface-patterned perm-selective nanoporous film like Nafion in single, open microchannels. Such a surface-patterned Nafion film can rapidly transport only cations from the anodic side to the cathodic side through the nanopore clusters so that it is possible to generate an ICP phenomenon near the Nafion film. In this work, we characterize transport phenomena and distributions of ion concentration under various electric fields near the Nafion film and show that single-channel based ICP (SC-ICP) is affected by Nafion film thicknesses, strengths of applied electric fields, and ionic strengths of buffer solutions. We also emphasize that SC-ICP devices have several advantages over previous dual-channel ICP (DC-ICP) devices: easy and simple fabrication processes, inherently leak-tight, simple experimental setup requiring only one pair of electrodes, stable and robust ICP induced rapidly, and low electrical resistances helping to avoid Joule heating, and membrane perm-selectivity breakdown but allowing as high bulk flow as an open, plain microchannel. As an example of applications, we demonstrate that SC-ICP devices not only have high potential in pre-concentrating proteins in massively parallel microchannels but also enable the concentration and lysis of bacterial cells simultaneously and continuously on a chip; therefore, proteins within the cells are extracted, separated from the concentrated cells and then pre-concentrated at a different location that is closer to the Nafion film. Hence, we believe that the SC-ICP devices have higher possibilities of being easily integrated with traditional microfluidic systems for analytical and biotechnological applications.

Lab-chip HPLC with integrated droplet-based microfluidics for separation and high frequency compartmentalisation.

We demonstrate the integration of a droplet-based microfluidic device with high performance liquid chromatography (HPLC) in a monolithic format. Sequential operations of separation, compartmentalisation and concentration counter were conducted on a monolithic chip. This describes the use of droplet-based microfluidics for the preservation of chromatographic separations, and its potential application as a high frequency fraction collector.

Protein separation using preparative-scale dynamic field gradient focusing.

Dynamic field gradient focusing uses an electric field gradient to separate and concentrate proteins in native buffers. A prototype preparative-scale dynamic field gradient focusing apparatus reproducibly separated hemoglobin and bovine serum albumin with a mean resolution of 2.64+/-0.503. Run-to-run variations in the hemoglobin's focal point and peak width appeared to be related to fluctuations in the shape of the electric field, rather than the 5% accuracy of the pump that provided the counter-flow in the separation annulus. The variation in the electric field gradient was probably due to the formation and expansion of an ion-depleted region at the top of the separation annulus.

Continuous-flow single-molecule CE with high detection efficiency.

This paper describes the use of two-beam line-confocal detection geometry for measuring the total mobility of individual molecules undergoing continuous-flow CE separation. High-sensitivity single-molecule confocal detection is usually performed with a diffraction limited focal spot (approximately 500 nm in diameter), which necessitates the use of nanometer-sized channels to ensure all molecules flow through the detection volume. To allow for the use of larger channels that are a few micrometers in width, we employed cylindrical optics to define a rectangular illumination area that is diffraction-limited (approximately 500 nm) in width, but a few micrometers in length to match the width of the microchannel. We present detailed studies that compare the performance of this line-confocal detection geometry with the more widely used point-confocal geometry. Overall, we found line-confocal detection to provide the highest combination of signal-to-background ratio and spatial detection efficiency when used with micrometer-sized channels. For example, in a 2 microm wide channel we achieved a 94% overall detection efficiency for single Alexa488 dye molecules when a 2 microm x 0.5 microm illumination area was used, but only 34% detection efficiency with a 0.5 microm-diameter detection spot. To carry out continuous-flow CE, we used two-beam fluorescent cross-correlation spectroscopy where the transit time of each molecule is determined by cross-correlating the fluorescence registered by two spatially offset line-confocal detectors. We successfully separated single molecules of FITC, FITC-tagged glutamate, and FITC-tagged glycine.

Characterization and performance of injection molded poly(methylmethacrylate) microchips for capillary electrophoresis.

Injection molded poly(methylmethacrylate) (IM-PMMA), chips were evaluated as potential candidates for capillary electrophoresis disposable chip applications. Mass production and usage of plastic microchips depends on chip-to-chip reproducibility and on analysis accuracy. Several important properties of IM-PMMA chips were considered: fabrication quality evaluated by environmental scanning electron microscope imaging, surface quality measurements, selected thermal/electrical properties as indicated by measurement of the current versus applied voltage (I-V) characteristic and the influence of channel surface treatments. Electroosmotic flow was also evaluated for untreated and O2 reactive ion etching (RIE) treated surface microchips. The performance characteristics of single lane plastic microchip capillary electrophoresis (MCE) separations were evaluated using a mixture of two dyes-fluorescein (FL) and fluorescein isothiocyanate (FITC). To overcome non-wettability of the native IM-PMMA surface, a modifier, polyethylene oxide was added to the buffer as a dynamic coating. Chip performance reproducibility was studied for chips with and without surface modification via the process of RIE with O2 and by varying the hole position for the reservoir in the cover plate or on the pattern side of the chip. Additionally, the importance of reconditioning steps to achieve optimal performance reproducibility was also examined. It was found that more reproducible quantitative results were obtained when normalized values of migration time, peak area and peak height of FL and FITC were used instead of actual measured parameters.

Nanoliter capillary electrochromatography columns based on collocated monolithic support structures molded in poly(dimethyl siloxane).

Following current trends in miniaturization of analytical chemistry, an inexpensive disposable analytical tool in the form of a liquid chromatography column fabricated on a poly(dimethyl siloxane) (PDMS) chip was created. Ease of fabricating the chromatography column was demonstrated by molding collocated monolithic support structures (COMOSS) directly in the column. Positive photo-resist, SPR 220, was used to create column structures on a negative relief master providing final channel dimensions of 2.7-5.2 microm wide by 10.0 microm deep, while monolithic dimensions were 9.8 x 9.8 x 10.0 microm - 12.3 x 12.3 x 10.0 microm. The ability to separate biological samples such as peptides from a tryptic digest of fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) was shown. Separations in capillary electrochromatographic (CEC) mode were performed yielding column efficiencies of 4.0 x 10(5) plates/m.

Partition and permeation of dextran in polyacrylamide gel.

Partition of sized FITC-dextrans in polyacrylamide gel showed a relationship between Kav and solute radius as predicted by the theory of Ogston, which is based solely on geometry of the spaces. Permeability data for the same dextrans were fit to several theories, including those based on geometry and those based on hydrodynamic interactions, and the gel structure predicted by the partition and permeability data were compared. The Brinkman effective-medium model (based on hydrodynamic interactions and requiring a measure of the hydraulic conductivity of the matrix) gave the best fit of permeability data with the values for fiber radius (rf) and void volume of the gel (epsilon) that were obtained from the partition data. The models based on geometry and the hydrodynamic screening model of Cukier, using the rf and epsilon from partition data, all predicted higher rates of permeation than observed experimentally, while the effective-medium model with added term for steric interaction predicted lower permeation than that observed. The size of cylindrical pores appropriate for the partition data predicted higher rates of permeation than observed. These relative results were unaffected by the method of estimating void volume of the gel. In sum, it appears that one can use data on partition of solute, combined with measurement of hydraulic conductivity, to predict solute permeation in polyacrylamide gel.